Identifying Pyroptosis-hub genes and immune infiltration in neonatal hypoxic-ischemic brain injury

Chi QinYue LiMeiying ChengHuixian LiRonghao MuJian JinBohao ZhangXin Zhao^*Xiaoan Zhang^*

• Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Background Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal brain injury and neurodevelopmental disorders. Pyroptosis, an inflammatory programmed cell death, may offer new therapeutic targets for HIE by modulating cytokine expression and related pathways. This study aims to identify HIE-associated pyroptosis genes and explore potential drugs and molecular mechanisms. Methods The gene microarray data of hypoxic-ischemic brain damage (HIBD) were obtained from the Gene Expression Omnibus (GEO) database. The Limma package was used to identify differentially expressed genes (DEGs). Weighted gene co - expression network analysis (WGCNA) was performed to find significant expression modules. GO and KEGG analyses were carried out for the pathway enrichment of DEGs, as well as protein–protein interaction (PPI) network analysis were subsequently conducted. Cytohubba software was employed to identify hub genes among DEGs. A random forest (RF) model assessed the pyroptosis-related genes, examining their diagnostic performance. Potential therapeutic drugs or compounds targeting the hub genes were screened through DSigDB, and their binding scores and affinities were evaluated by molecular docking. Results 96 DEGs with HIBD were identified in our result, including 89 up-regulated genes and 7 down-regulated genes. GO and KEGG results indicated that these DEGs were mostly enriched in Cytokine-cytokine receptor interaction, IL-17 signaling pathway and TNF signaling pathway. Using Cytoscape software and WGCNA-related modules, we identified three hub genes—Tnf, IL1B, and Tlr2—which were further validated in other transcriptomic datasets, all showing significant differential expression. Random forest analysis demonstrated that these three hub genes had AUC values > 0.75, indicating strong diagnostic performance. Immune infiltration analysis revealed that, compared to the control group, the HIBD group exhibited higher levels of innate immune cells (e.g., macrophages, M0 cells, and dendritic cells) and adaptive immune cells (e.g., CD8 naïve T cells, CD4 follicular helper T cells, and Th1 cells). The ssGSEA algorithm results indicated differences in 25 types of immune cells and 10 immune functions. The hub genes were also validated finally. Conclusion Tnf, Il1b and Tlr2 may be potential hub pyroptosis-related genes for HIBD. The results of this study could improve the understanding of the mechanisms underlying pyroptosis in HIBD.